Aneksyna A6, białko wiążące cholesterol i nukleotydy, uczestniczące w naprawie błon biologicznych i w transporcie pęcherzykowym

Joanna Bandorowicz-Pikuła

Agnieszka Kinga Seliga

Laboratory of Cellular Metabolism, Nencki In-stitute of Experimental Biology, 3 Pasteura St., 02-093 Warsaw, Poland

*_{e-mail: j.bandorowicz-pikula@nencki.gov.pl}

Received: September 25, 2018 Accepted: October 23, 2018

Key words: annexin A6, cholesterol

metabo-lism, membrane repair, vesicular transport

Acknowledgements: The research in Authors’

Laboratory is supported by statutory fund from the Nencki Institute of Experimental Bio-logy, Polish Academy of Sciences and by an Opus grant, reg. no 2016/23/B/NZ3/03116, from the National Science Center to JBP.

Annexin A6 as a cholesterol and nucleotide binding protein involved

in membrane repair and in controlling membrane transport

during endo- and exocytosis

ABSTRACT

A

INTRODUCTION – THE ANNEXINS

The vertebrate annexin (AnxA) superfamily consists of 12 members of

cal-cium and phospholipid binding proteins which share high structural homology

[Morgan et al. 2004; 2006; Gerke et al. 2005; Bandorowicz-Pikula 2003;

Bandoro-wicz Pikula et al. 2001; 2012; Domon et al. 2012; Kodavali et al. 2014; Grewal et al.

2016]. In keeping with this hallmark feature, annexins have been implicated in

Ca

_{-controlled regulation of a broad range of membrane related events, which}

may suggest its potential therapeutic value, namely, the regulation of immune

response and control of tissue homeostasis [Schloer et al. 2018]. Recent highlights

concerning transgenic (knockout animals) are summarized in table 1.

In this review, we focus on one of the main subjects of our investigations,

annexin A6 (AnxA6), its properties and potential functions in addition to major

achievements made by us in this field since 1997, we discuss recent advances

related to hypothetical functions of AnxA6, including organization of biological

membranes, or membrane repair mechanisms. The latter may be controlled in

response to a disrupted cellular hemostasis and vesicle-related cellular

process-es, such as biomineralization [Balcerzak et al. 2008; Kapustin & Shanahan 2016;

Minashima & Kirsch 2018; Bottini et al. 2018], transport and storage of cholesterol

[Enrich et al. 2011; 2017; Reverter et al. 2011].

ANNEXIN A6 AT THE NENCKI INSTITUTE

Annexins were first introduced to the Nencki Institute as one among

sever-al experimentsever-al subjects of the Laboratory of Plasma Membrane Receptors at

early 1990s [Bandorowicz et al. 1992; 1996; Sobota et al. 1993], and since 1997

further explored at the Laboratory of Lipid Biochemistry and Laboratory of

Cellular Metabolism, but also since 1991 by the members of Laboratory of

Cal-cium Binding Proteins [Filipek et al. 1991; 1995; Filipek & Wojda 1996]. During

this time the important discoveries have been made concerning the annexin

structure, potential ligands and finally their cellular and organismal functions.

They are listed in table 2. It should be underlined that structural features,

bio-chemical and biophysical properties of AnxA6 together with its functions have

been well characterized in vitro and in cellular systems mainly on the basis of

two fundamental discoveries – nucleotide binding properties of AnxA6

[Ban-dorowicz-Pikula & Awasthi 1997; Ban[Ban-dorowicz-Pikula et al. 1997a; 1997b; 1999;

Bandorowicz-Pikula 1998; Bandorowicz-Pikula & Pikula 1998; Danieluk et al.

1999] and its interaction with cholesterol and cholesterol enriched biological

membranes in a calcium- and pH-dependent manner [Sztolsztener et al. 2010;

2012; Domon et al. 2010; 2011; 2013a,b]. It should be stressed that cholesterol

binding properties of AnxA6, characterized by us on the basis of a series of

in vivo and ex vivo experiments, have been extensively studied in many other

laboratories around the world. The obtained results suggest that AnxA6 acts

as a multifunctional scaffold protein and is able to recruit vast number of

sig-naling proteins, modulates cholesterol transport and its

distribution within the cell, and also regulates membrane

transport through actin dynamics. These activities suggest

that AnxA6 may contribute to the formation of specific

protein complexes and membrane domains relevant in

sig-nal transduction, cholesterol homeostasis and

endo-/exo-cytosis [Grewal et al. 2017].

MEMBRANE-RELATED FUNCTIONS OF ANNEXIN A6

MEBRANE LATERAL ORGANIZATION

Annexin A6, as a cholesterol binding and

multifunc-tional scaffold protein plays a crucial role in cell motility

[Hayes et al. 2004; Monastyrskaya et al. 2009; Grewal et al.

2017] and is implicated also in cell signaling [Koese et al.

2013; Hoque et al. 2014; Qi et al. 2015; Cornely et al. 2016;

Raouf et al. 2018]. Moreover, annexin A6 has been

report-ed to regulate a formation of multifunctional signaling

complexes at the membranes, affect membrane lateral

or-ganization, or influence cholesterol metabolism and

dis-tribution, but also to participate in the vesicular transport

both in endo- and exocytosis [Cubells et al. 2007; 2008;

Enrich et al. 2014; Garcia-Melero et al. 2016; Cairns et al.

2017]. In addition, it has been shown that AnxA6

recruit-ed to the plasma membrane is able to affect membrane

re-modelling, e.g. upregulated AnxA6 in the cell decreased

plasma membrane order through the regulation of

cellu-lar cholesterol homeostasis and its interaction with the

actin cytoskeleton in the living cells [Alvarez-Guaita et

al. 2015]. Strong experimental evidence has been

accu-mulated that AnxA6 due to its unique, among annexins,

structure affecting the distribution of cell specific surface

receptors, recruits the interaction partners and

simultane-ously bridges specialized membrane domains with

corti-cal actin surrounding activated receptors [Cornely et al.

2011].

MEBRANE REPAIR

The features of AnxA6 described above may be further

extended to the observations suggesting its participation

Table 1. Properties of mammalian annexins revealed on the basis of analyses of knockout animals.

Annexin Gene _encoding MW (Da) Total aa C-terminal core domain Some proposed functions*

Annexin A1 ANXA1 38,714 346 4 repeat domains anti- or pro-inflammatory responses, wound closure, epithelial motility, cancer cell metastasis, insulin secretion, cell fusion, vesicular transport, cell signaling, uptake of viruses

Annexin A2 ANXA2 40,41138,604 357₃₃₉ 4 repeat domains cancer cell metastasis, fibrinolysis, pathogen _{recognition, defense against bacterial infection} Annexin A3 ANXA3 36,375 323 4 repeat domains nd

Annexin A4 ANXA4 35,883 321 4 repeat domains cardiomyocyte signaling, integrity of urothelium Annexin A5 ANXA5 35,937 320 4 repeat domains biomineralization, thrombosis, angiogenesis, _{recognition of apoptotic cells}

Annexin A6 ANXA6 75,873_72,423 673₆₄₁ 8 repeat domains _{and linker} calcium homeostasis, plasma membrane organization, membrane repair, gluconeogenesis, biomineralization, chondrocyte differentiation Annexin A7 ANXA8 52,739_50,316 488₄₆₆ 4 repeat domains cardiac contraction and remodeling, insulin _{secretion, cell proliferation} Annexin A8 ANXA9 36,881 327 4 repeat domains nd

Annexin A9 ANXA10 38,364 345 4 repeat domains nd Annexin A10 ANXA11 37,278 324 4 repeat domains nd Annexin A11 ANXA12 54,390 505 4 repeat domains nd Annexin A13 ANXA13 35,415_39,744 316₃₅₇ 4 repeat domains nd

Information taken from https://www.uniprot.org, www.ncbi.nlm.nih.gov/protein/. *Some intra and extracellular functions suggested on the basis of the experiments performed using knockout animals, reviewed in [Grewal

in membrane repair mechanisms. Efficient cell membrane

repair mechanisms are essential for maintaining

mem-brane integrity and, thus, for cell life [Lauritzen et al.

2015; Demonbreun et al. 2016; Boye et al. 2017].

Initially, it has been observed that AnxA1 is involved

in the repair of plasmalemmal lesions induced by

bacteri-al toxins. Furthermore, highly Ca

_{-sensitive AnxA6, that}

responds faster to [Ca

_]

i

elevation than AnxA1, promotes

formation of lesions, and therefore is able to react to a

limited and sustained membrane injury [Potez et al. 2011].

The AnxA6 contribution to membrane repair mechanism

has been further elaborated on the basis of in vitro

obser-vations that AnxA4 and AnxA6 involved in plasma

mem-brane repair cause rapid closure of micron-size holes in

membranes. It has been demonstrated that AnxA4 binds

to membranes and generates curvature force, whereas

AnxA6 induces constriction force. In cells, plasma

mem-brane injury and concomitant Ca

_{influx result in AnxA4}

recruitment to the vicinity of membrane wound edges.

Then, homo-trimerization of AnxA4 leads to membrane

curvature near the edges. Mediated by AnxA6

constric-tion force is responsible for pulling the wound edges

to-gether for membrane fusion and final repair [Boye et al.

2017]. In agreement are observations performed by means

of whole genome sequencing and RNA sequencing which

identified AnxA6 on the mouse model of muscular

dys-trophy associated with cardiomyopathy. Its truncated

version called ANXA6N32 was found to be responsible

for disrupting the whole AnxA6-rich cap and the

associ-ated (surrounding) repair zone at the site of sarcolemma

disruption, resulting in a membrane leak, characteristic

for muscular dystrophy [Swaggart et al. 2014].

VESICULAR TRANSPORT

AnxA6 features allowed many investigators to think

about this protein as a potential modulator of vesicular

transport events. It has been suggested that AnxA6 is

impli-cated in endocytosis, especially at the stage of fusion of

au-tophagosomes with endocytic compartment in hepatocytes

[Tebar et al. 2014; Enrich et al. 2017]. Moreover, AnxA6

high-ly expressed in smooth muscles, hepatocytes, endothelial

cells and cardiomyocytes, has been found to affect various

stages of endocytotic route of cholesterol transport [Cubells

et al. 2007; Enrich et al. 2011; Reverter et al. 2011; Rentero et

al. 2018].

In addition, AnxA6 was found to participate in

choles-terol storage and the control of late endosomal cholescholes-terol

levels, that modulate integrin recycling and cell migration

[Garcia-Melero et al. 2018], as well as influenza A

replica-tion and propagareplica-tion [Musiol et al. 2013]. AnxA6 has also

been linked to triglyceride storage in adipocytes [Cairns et

al. 2017].

Participation of annexins in exocytosis was first

postu-lated almost 30 years ago [Creutz 1992]. Further studies

has revealed, that the number of observations suggesting

functioning of AnxA6 in exocytosis is limited.

Investiga-tors, however, agree that this multifunctional protein plays

a regulatory role in membrane trafficking during exocytosis

too [Enrich et al. 2017; Cairns et al. 2018].

CONCLUDING REMARKS

We have actively contributed to experiments, results

of which show that AnxA6 is an exceptional member

Table 2. Properties and potential cellular functions of annexin A6 studied in the Laboratory of Lipid Biochemistry.

Feature Description References

Cellular localization Plasma membrane, lysosomes and endosomes, matrix vesicles

[Bandorowicz et al. 1992; Balcerzak

et al. 2008; Strzelecka-Kiliszek et al.

2008; Sztolsztener et al. 2010; 2012; Cmoch et al. 2011; Bottini et al. 2018]

Membrane binding properties and ion channel-like activity

Calcium dependent binding to plasma membrane phosphatidylserine

A pH and calcium dependent interaction of AnxA6 with cholesterol, identification of cholesterol binding domain in AnxA6

Mechanism of folding of AnxA6 in membranes at acidic pH

[Bandorowicz-Pikula et al. 1996] [Domon et al. 2010; 2011; 2013a; 2013b] [Golczak et al. 2001; 2004; Pikula 2003; Buzhynskyy et al. 2009]

Nucleotide binding properties Identification of a putative consensus sequence for the nucleotide-binding site in AnxA6 GTP-induced ion channel activity of AnxA6

[Kirilenko et al. 2002; 2006; Bandorowicz-Pikula 2003; Bandorowicz-Bandorowicz-Pikula et al. 2003]

Intracellular functions

Lateral organization of plasma membrane – microdomains Transport and storage of cholesterol

Catecholamine and interleukin-2 secretion Biomineralization and matrix vesicles biogenesis

[Domon et al. 2012]

[Bandorowicz-Pikula et al. 2012] [Podszywalow-Bartnicka et al. 2007; 2010; Strzelecka-Kiliszek et al. 2008] Pathogenesis Lipid storage in Niemann-Pick type C disease _{associated with mitochondrial dysfunction} [Wos et al. 2016; 2018]

of the annexin family of proteins resembling genuine

cholesterol-interacting proteins. Our studies indicate

particularly that AnxA6 intracellular localization and

membrane binding at low pH is determined by

choles-terol. Although, the overall picture of possible AnxA6

functions still requires further studies to

identify/clari-fy/completely unveil physiological and/or pathological

processes involving AnxA6 ability to change membrane

permeability to ions or mechanisms of membrane repair.

To sum up, step should be taken to elucidate the overall

importance of AnxA6 for the whole organism as it may

form specific target to identify and cure human diseases

in which AnxA6 may play a significant role.

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Aneksyna A6, białko wiążące cholesterol i nukleotydy, uczestniczące

w naprawie błon biologicznych i w transporcie pęcherzykowym

Joanna Bandorowicz-Pikuła

, Agnieszka Kinga Seliga

Pracownia Metabolizmu Komórki, Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN, ul. Pasteura 3, 02-093 Warszawa

*e-mail: j.bandorowicz-pikula@nencki.gov.pl

Słowa kluczowe: aneksyna A6, cholesterol, naprawa błon biologicznych, transport pęcherzykowy

STRESZCZENIE

Aneksyny, rodzina białek wiążących jony wapnia i błony biologiczne, były badane w Instytucie Biologii Doświadczalnej im. Marcelego Nenckiego w Warszawie od wczesnych lat 90. XX wieku. Szczególną uwagę poświęcono strukturze aneksyn, potencjalnym ligandom tych białek oraz ich funkcji, np. w procesie biomineralizacji zachodzącym w normie i w stanach patologicznych. Wyniki badań prowadzonych w wielu laboratoriach na świecie wskazują, że aneksyny odgrywają bardzo ważną rolę w organizacji błon biologicznych. W tym artykule prze-glądowym opisujemy jednego z największych pod względem masy cząsteczkowej przedstawiciela rodziny aneksyn, aneksynę A6 (AnxA6), białko wykazujące zdolność wiązania się z cholesterolem i nukleotydami oraz zmieniające przepuszczalność błony dla jonów w odpowiedzi na obniżenie wewnątrzkomórkowego pH. Dodatkowo, opisano funkcje AnxA6, takie jak udział w tworzeniu mikrodomen błonowych, w naprawie uszkodzeń błony plazmatycznej oraz w regulacji transportu pęcherzykowego.

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Woś M, Szczepanowska J, Pikuła S, Tylki-Szymańska A, Zabłocki K, Bandorowicz-Pikuła J (2016) Mitochondrial dysfunction in fibroblasts derived from patients with Niemann-Pick type C disease. Arch Bio-chem Biophys 593: 50-59

Woś M, Komiażyk M, Pikuła S, Tylki-Szymańska A, Bandorowicz-Pikuła J (2018) Activation of mTOR kinase and GSK-3β accompanies abnor-mal accumulation of cholesterol in fibroblasts from Niemann-Pick type C patients. J Cell Biochem in press